Use of Flubendazole and Vinca Alkaloids for Treatment of Hematological Diseases

ABSTRACT

Provided are methods for treating a hematological malignancy comprising administering an effective amount of flubendazole alone or in combination with a vinca alkaloid. Also provided are compositions and kits comprising an effective amount of flubendazole and/or a vinca alkaloid for use in the methods of the disclosure.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a PCT Patent Application, which claims the prioritybenefit of U.S. Provisional Patent Application No. 61/250,303, filedOct. 9, 2009, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to methods and uses of flubendazole and vincaalkaloids for treating hematological malignancies and to compositionscomprising flubendazole and vinca alkaloids.

BACKGROUND OF THE DISCLOSURE

Flubendazole has been extensively evaluated in humans and animals forthe treatment of intestinal parasites as well as for the treatment ofsystemic worm infections. In these studies, patients have received up to50 mg/kg orally daily for 24 months without serious adverseeffects.¹²⁻¹⁴ Healthy volunteers have also received single oral doses upto 2000 mg without toxicity.¹⁵ In sheep receiving a single flubendazoledose of 5 mg/kg intravenously, no toxicity was observed and the areaunder the curve (AUC) was 6.53 μg·h/mL (22 μM).¹⁶

Vinca alkaloids are currently used in the treatment of leukemia andmyeloma, and neurotoxicity is a dose limiting toxicity of vincristine.

SUMMARY OF THE DISCLOSURE

An aspect of the disclosure includes a method of treating ahematological malignancy in a subject in need thereof comprisingadministering to the subject an effective amount of flubendazole and/ora pharmaceutically acceptable solvate and/or prodrug thereof.

Another aspect of the disclosure includes a method of treating ahematological malignancy in a subject in need thereof comprisingadministering to the subject an effective amount of flubendazole and/ora pharmaceutically acceptable solvate and/or prodrug thereof, and aneffective amount of a vinca alkaloid and/or a pharmaceuticallyacceptable salt, solvate and/or prodrug thereof.

In an embodiment, the vinca alkaloid is selected from vinblastine,vincristine, vindesine and/or vinolrebine and pharmaceuticallyacceptable salts, solvates and/or prodrugs thereof.

In another embodiment, the hematological malignancy is drug resistant toa vinca alkaloid and/or overexpresses P glycoprotein (Pgp).

In a further embodiment, the hematological malignancy is a leukemia,lymphoma or myeloma.

In yet another embodiment, the flubendazole and/or a pharmaceuticallyacceptable solvate and/or prodrug thereof and/or the vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof are comprised in a single oral dosage form or separate oraldosage forms.

In another embodiment, the flubendazole and/or a pharmaceuticallyacceptable solvate and/or prodrug thereof and/or the vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof are comprised in a single intravenous dosage form or separateintravenous dosage forms.

In another aspect, the disclosure includes use of an effective amount offlubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof for treating a hematological malignancy.

In an embodiment, the disclosure includes use of an effective amount offlubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof in combination with an effective amount of a vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof for treating a hematological malignancy.

A further aspect includes use of an effective amount of flubendazoleand/or a pharmaceutically acceptable solvate and/or prodrug thereof forthe manufacture of a medicament for treating a hematological malignancy.

In an embodiment, the disclosure includes use of an effective amount offlubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof in combination with an effective amount of a vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof for the manufacture of a medicament for treating a hematologicalmalignancy.

Another aspect of the disclosure includes a composition comprising aneffective amount of flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof and a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof.

In an embodiment, the disclosure includes a composition comprising aneffective amount of flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof and optionally a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof fortreating a hematological malignancy.

A further aspect includes a kit comprising flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof andoptionally a vinca alkaloid and/or a pharmaceutically acceptable salt,solvate and/or prodrug thereof for treating a hematological malignancy.In an embodiment, the kit comprises flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof andinstructions for combination use with a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof fortreating a hematological malignancy.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Flubendazole induces cell death in malignant cell lines

(A) OCI-AML2 cells were treated for 72 h with increasing concentrationsof benzimidazoles. After incubation, cell growth and viability wasmeasured by the MTS assay. Data represent the mean percentage of viablecells ±SD from a representative experiment.

(B) Leukemia, lymphoma and myeloma cell lines were treated withincreasing concentrations of flubendazole. Seventy two hours afterincubation, cell growth and viability was measured by the MTS assay.Data represent the mean percentage of viable cells ±SD fromrepresentative experiments.

(C) Primary AML cell samples (n=3) were plated in a methylcellulosecolony forming assay with increasing concentrations of flubendazole.Colonies were counted 7 days after plating and normalized to culturestreated with buffer alone. Data represent the mean±SD from 3 independentexperiments performed in duplicate.

FIG. 2: Flubendazole delays tumor growth and reduces tumor weight inleukemia and myeloma mouse xenografts

Sub-lethally irradiated SCID mice were injected subcutaneously withOCI-AML2 cells (n=20; 10 per group). After implantation, mice weretreated with 50 mg/kg flubendazole (A), 20 mg/kg flubendazole (B), orvehicle control by intraperitoneal injection daily. Tumor volume wasmeasured over time. After 16 days (20 mg/kg dose) or 18 days (50 mg/kgdose), mice were sacrificed and tumors were excised, measured andweighted.

(C) Sub-lethally irradiated SCID mice were injected subcutaneously withOPM2 cells (n=20; 10 per group). One week after implantation, whentumors were palpable, mice were treated with 50 mg/kg flubendazole orvehicle control by intraperitoneal injection twice daily. Tumor volumeand body weight was measured over time. After 17 days, mice weresacrificed and tumors were excised, measured and weighted.

Data are presented as means±SEM. Differences in tumor volume and tumorweight were analyzed by an unpaired t-test: ***p<0.0001; **p<0.001;*p<0.05.

FIG. 3: Flubendazole inhibits tubulin structure, polymerization andfunction

(A) Flubendazole (100 μM) and vinblastine (10 μM) were incubated withbovine tubulin (1.5 μM) and the conformational changes were monitoredspectrophotometrically by measuring the decrease in the number ofreactive cysteine residues at an absorbance of 412 nm as described inthe materials and methods. A representative figure is shown.

(B) Flubendazole (100 μM), colchicine (6.0 μM) and taxol (6.0 μM) wereincubated with bovine tubulin (1.8 mg/ml) and the effects onpolymerization were monitored spectrophotometrically by measuringturbidity at 340 nm as described in the materials and methods. Arepresentative figure is shown.

(C) Tubulin (5.0 μM) was incubated for 30 min with 100 μM vinblastine,100 μM flubendazole, or buffer control. After incubation, colchicine (10μM) was added and incubated for 60 minutes. Fluorescence of thetubulin-colchicine complex was measured with excitation and emissionwavelengths of 360 nm and 430 nm, respectively. Reduced fluorescenceindicates binding at the colchicine site. *p<0.01 (ANOVA, bonferoni posthoc). A representative figure is shown.

(D) PPC-1 cells were treated with 1.0 μM flubendazole (i) or control(ii) for 24 h and stained with DAPI and an anti α-tubulin AlexaFluor 488nm antibody. Images were captured using an Olympus Fluorview confocalmicroscope at room temperature. Representative confocal micrographs at40× are shown.

(E) HeLa cells were grown to confluence and a wound created on the cellmonolayer using a 200 μL pipette. Cells were treated with increasingconcentrations of flubendazole and imaged every 2 h for 8 h. Woundhealing was measured as described in the materials and methods.Representative data is shown and are presented as % wound recovery.*p<0.05 (ANOVA, bonferroni post hoc).

FIG. 4: Flubendazole induces cell cycle arrest and mitotic catastrophe

OCI-AML2 cells (A) or PPC-1 cells (B) were incubated with 1.0 μMflubendazole or buffer control for 24 h. Cells were then stained with PIand the DNA content was measured by flow cytometry. A representativefigure is shown.

(C) PPC-1 cells were treated as above and stained with anti α-tubulinantibody and DAPI, as described in the materials and methods. Cells wereimaged by confocal microscopy and the number of multi-nucleated cellswas enumerated. Data represent the mean±SD percent of multinucleatedcells from a representative experiment *p<0.0001 (unpaired t test).Insert: a representative multi-nucleated cell.

FIG. 5: Flubendazole synergizes with vinblastine and vincristine

The effects of increasing concentrations of flubendazole in combinationwith vinblastine (A) or colchicine (B) on the viability of OCI-AML2cells. Cell viability was measured by the MTS assay after 72 hincubation. Data were analyzed with Calcusyn software as described in‘Materials and Methods’. Combination index (CI) versus Fractional effect(Fa) plot showing the effect of the combination of flubendazole andvinblastine or colchicine. CI<1 indicates synergism. One of tworepresentative isobologram experiments performed in triplicate is shown.

Sub-lethally irradiated SCID mice were injected subcutaneously withOCI-AML2 cells (n=40; 10 per group). After implantation, mice weretreated with (C) 15 mg/kg flubendazole, 0.3 mg/kg vinblastine, acombination of flubendazole and vinblastine, or vehicle control; or (D)20 mg/kg flubendazole, 0.25 mg/kg vincristine, a combination offlubendazole and vincristine, or vehicle control. After 16 (C) or 18 (D)days, mice were sacrificed and tumors were excised, measured andweighted. Data represent the mean±SD tumor weight. A representativeexperiment is shown. *p<0.05, *p<0.01, **p<0.001 (Unpaired t-test).

FIG. 6: Flubendazole does not alter glucose uptake

(A) OCI-AML2 cells were incubated with increasing concentrations offlubendazole for 16 h and the uptake of 3H-deoxy-D-glucose was measuredas in the materials and methods. Data represent the mean±SD glucoseuptake. A representative experiment is shown.

(B) OCI-AML2 cells were grown in media supplemented with increasingconcentrations of glucose in the presence or absence of 1.0 μMflubendazole for 72 h. After incubation, cell growth and viability wasmeasured by the MTS assay. Data represent the mean±SD percent viablecells. A representative experiment is shown.

FIG. 7: Drug treatments do not affect mouse body weight

(A) Mice injected with flubendazole (20 mg/kg), vincristine (0.25 mg/kg)or the combination of flubendazole and vincristine does not affect mousebody weight after 18 days of treatment. (B) Mice injected withflubendazole (15 mg/kg), vinblastine (0.3 mg/kg) or the combination offlubendazole and vinblastine does not affect mouse body weight after 16days of treatment.

DETAILED DESCRIPTION OF THE INVENTION

Drugs with previously unrecognized anti-cancer activity could be rapidlyrepurposed for this new indication given their prior toxicity testing.To identify such compounds, chemical screens were conducted whichidentified the antihelmintic flubendazole. Flubendazole induced celldeath in leukemia and myeloma cell lines and primary patient samples atnanomolar concentrations. Moreover, it delayed tumor growth in leukemiaand myeloma xenografts without evidence of toxicity. Mechanistically,flubendazole inhibited tubulin polymerization by binding tubulin at asite distinct from vinblastine. In addition, cells resistant tovinblastine due to over-expression of p-glycoprotein (Pgp) remainedfully sensitive to flubendazole, indicating that flubendazole canovercome some forms of vinblastine resistance. Given the differentmechanisms of action, the combination of flubendazole and vinblastinewas evaluated in vitro and in vivo. Flubendazole synergized withvinblastine to reduce the viability of OCI-AML2 cells. In addition,combinations of flubendazole with vinblastine or vincristine in aleukemia xenograft model delayed tumor growth more than either drugalone. Therefore, flubendazole is a novel microtubule inhibitor thatdisplays therapeutic activity in leukemia and myeloma. Given its priorsafety record when evaluated for the treatment of gastrointestinalparasites, flubendazole can be repurposed for the treatment of patientswith hematologic malignancies.

I. DEFINITIONS

The term “flubendazole” as used herein refers to a compound having theformula:

or a pharmaceutically acceptable solvate or prodrug thereof.

Flubendazole has been sold under the brand names Flubenol®, Biovermin®,Flubenol KH®, Flumoxal® and Flutelmium®. Methods of making flubendazoleare known in the art and described for example in U.S. Pat. No.3,657,267, herein incorporated by reference.

The term “vinca alkaloids” as used herein refers to a group ofantimitotic chemotherapeutic drugs that can be isolated from theperiwinkle plant (Vinca rosea) and includes without limitation,vinblastine, vincristine, vindesine and vinorelbine as well as mixturesthereof.

The term “vinblastine”, also known as “vincaleukoblastine”, as usedherein refers a compound having the formula:

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Vinblastine can be isolated or synthesized using methods known in theart, for example using methods described in U.S. Pat. No. 5,034,320.Pharmaceutically acceptable salts include for example vinblastinesulfate. Vinblastine is available for example under the brand nameVelbe®.

The term “vincristine”, also known as “leurocristine”, as used hereinrefers to a compound having the formula:

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Vincristine can be isolated or synthesized using methods known in theart, for example as described in U.S. Pat. No. 4,767,855, and referencescited therein. Pharmaceutically acceptable salts include for examplevincristine sulfate. Vincristine is available for example under thebrand name Oncovin®.

The term “vindesine” as used herein refers to a compound having theformula:

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Vindesine can be isolated or synthesized using methods known in the artincluding for example as described in U.S. Pat. No. 4,210,584 andreferences cited therein. Pharmaceutically acceptable salts include forexample vindesine sulfate. Vindesine is sold for example under the brandname Eldisine®.

The term “vinorelbine” as used herein refers to a compound having theformula:

or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Vinolrelbine can be isolated or synthesized using methods known in theart including for example as described in Fahy, J. et al. Biorg. Med.Chem. Lett. 2002, 12:505-507. Pharmaceutically salts include for examplevinolrebine tartrate. Vinolrebine is sold for example under the brandname Navelbine®.

The term “cell death” as used herein includes all forms of cell deathincluding for example necrosis and apoptosis.

The term “pharmaceutically acceptable salt” means an acid or basicaddition salt, which is suitable for or compatible with the treatment ofpatients.

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any basic compound.Basic compounds that form an acid addition salt include, for example,compound comprising an amine group. Illustrative inorganic acids whichform suitable salts include hydrochloric, hydrobromic, sulfuric andphosphoric acids, as well as metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids that form suitable salts include mono-, di-, and tricarboxylicacids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic,phenylacetic, cinnamic and salicylic acids, as well as sulfonic acidssuch as p-toluene sulfonic and methanesulfonic acids. Either the mono ordi-acid salts can be formed, and such salts may exist in either ahydrated, solvated or substantially anhydrous form. In general, acidaddition salts are more soluble in water and various hydrophilic organicsolvents, and generally demonstrate higher melting points in comparisonto their free base forms. The selection of the appropriate salt will beknown to one skilled in the art.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acidic compound. Acidic compounds that form a basic addition saltinclude, for example, compounds comprising a carboxylic acid group.Illustrative inorganic bases which form suitable salts include lithium,sodium, potassium, calcium, magnesium or barium hydroxide. Illustrativeorganic bases which form suitable salts include aliphatic, alicyclic oraromatic organic amines such as methylamine, trimethylamine andpicoline, alkylammonias or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The term “solvate” as used herein means a compound or itspharmaceutically acceptable salt, wherein molecules of a suitablesolvent are incorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”. The formation ofsolvates will vary depending on the compound and the solvate. Ingeneral, solvates are formed by dissolving the compound in theappropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions.

In general, prodrugs will be functional derivatives of parent compoundswhich are readily convertible in vivo into the parent compound fromwhich it is notionally derived. Prodrugs include, for example,conventional esters formed with an available, carboxylic acid, hydroxyand/or amino group. For example, available OH and/or NH₂ groups in acompounds is acylated using an activated acid in the presence of a base,and optionally, in inert solvent (e.g. an acid chloride in pyridine).Some common esters which have been utilized as prodrugs are phenylesters, aliphatic (C₈-C₂₄) esters, acyloxymethyl esters, carbamates andamino acid esters. In certain instances, prodrugs are those in which thecarboxylic acid, hydroxy and/or amino groups in a compound is masked asa group which can be converted to carboxylic acid, hydroxy and/or aminogroups in vivo. Conventional procedures for the selection andpreparation of suitable prodrugs are described, for example, in “Designof Prodrugs” ed. H. Bundgaard, Elsevier, 1985.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example in thecontext or treating a hematological malignancy, an effective amount isan amount that for example induces remission, reduces tumor burden,and/or prevents tumor spread or growth compared to the response obtainedwithout administration of the compound(s). Effective amounts may varyaccording to factors such as the disease state, age, sex, weight of thesubject. The amount of a given compound that will correspond to such anamount will vary depending upon various factors, such as the given drugor compound, the pharmaceutical formulation, the route ofadministration, the type of disease or disorder, the identity of thesubject or host being treated, and the like, but can nevertheless beroutinely determined by one skilled in the art.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with early stagemultiple myeloma is treated to prevent progression or alternatively asubject in remission is treated to prevent recurrence. Treatment methodscomprise administering to a subject a therapeutically effective amountof one or more compounds or compositions described in the presentdisclosure and optionally consists of a single administration, oralternatively comprises a series of applications. For example, thecompounds or compositions described herein are administered at leastonce a week, from about one time per week to about once daily to aboutfour times daily for a given treatment. The length of the treatmentperiod depends on a variety of factors, such as the severity of thedisease, the age of the patient, the concentration, the activity of thecompound(s), and/or a combination thereof. It will also be appreciatedthat the effective dosage of the compound used for the treatment orprophylaxis may increase or decrease over the course of a particulartreatment or prophylaxis regime. Changes in dosage may result and becomeapparent by standard diagnostic assays known in the art. In someinstances, chronic administration may be required.

The dosage administered will vary depending on the use and known factorssuch as the pharmacodynamic characteristics of the particular compound,and its mode and route of administration, age, health, and weight of theindividual recipient, nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Dosage regimemay be adjusted to provide the optimum therapeutic response.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and suitably refers to humans.

The term “hematological malignancy” or “hematological cancer” as usedherein refers to cancers that affect blood and bone marrow.

The term “hematological cancer cell” as used herein refers a cancerouscell of the blood and bone marrow lineages, including primary cells.Hematological cancer cells include for example leukemia cells such asleukemia cells represented by CEM, TEX, THP1, HL-60, RSV411, K562,Jurkat, U937, OCI-M2, OCI-AML2 and NB4 leukemia cell lines and cellsphenotypically similar thereto, lymphoma cells such as lymphoma cellsrepresented MDAY-D2 and cell phenotypically similar thereto, andmultiple myeloma cells such as multiple myeloma cells represented byOPM2, KMS11, LP1, UTMC2, KSM18, KSM12, H929, JJN3 and OCIMy5 myelomacell lines and cells phenotypically similar thereto. Hematologicalcancer cells also include chronic myelogenous leukemia cells, includingcells representing the blast crises phases such as K562 and cellsphenotypically similar thereto; AML cells such as represented by HL-60,K562, OCI-M2, and NB4 and cells phenotypically similar thereto, ALLcells such as represented by RSV411 and Jurkat and cells phenotypicallysimilar thereto, and lymphoma cells such as represented by MDAY-D2 andcells phenotypically similar thereto.

The term “leukemia” as used herein means any disease involving theprogressive proliferation of abnormal leukocytes found in hemopoietictissues, other organs and usually in the blood in increased numbers. Forexample, leukemia includes acute myeloid leukemia (AML), acutelymphocytic leukemia (ALL) and chronic myelogenous leukemia (CML).

The term “lymphoma” as used herein means any disease involving theprogressive proliferation of abnormal lymphoid cells. For example,lymphoma includes mantle cell lymphoma, Non-Hodgkin's lymphoma, andHodgkin's lymphoma. Non-Hodgkin's lymphoma would include indolent andaggressive Non-Hodgkin's lymphoma. Aggressive Non-Hodgkin's lymphomawould include intermediate and high grade lymphoma. IndolentNon-Hodgkin's lymphoma would include low grade lymphomas.

The term “myeloma” and/or “multiple myeloma” as used herein means anytumor or cancer composed of cells derived from the hemopoietic tissuesof the bone marrow. Multiple myeloma is also knows as MM and/or plasmacell myeloma.

The term “phenotypically similar” refers to a cell type that exhibitsmorphological, physiological and/or biochemical characteristics similarto another cell type. For example, a cell that is phenotypically similarto an AML cell can include a cell that comprises Auer rods. As anotherexample, U937 cells which are derived from a patient with lymphoma, showmorphological similarity to monocytoid AML cells. As a further examplethe leukemia cell line NB4 differentiates similar to promyelocytic cells(PML) with all trans retinoic acid (ATRA) and thereby represents a“phenotypically similar” model of PML cells.

As used herein, “contemporaneous administration” and “administeredcontemporaneously” means that two substances are administered to asubject such that they are both biologically active in the subject atthe same time. The exact details of the administration will depend onthe pharmacokinetics of the two substances in the presence of eachother, and can include administering one substance within 24 hours ofadministration of the other, if the pharmacokinetics are suitable.Designs of suitable dosing regimens are routine for one skilled in theart. In particular embodiments, two substances will be administeredsubstantially simultaneously, i.e. within minutes of each other, or in asingle composition that comprises both substances.

The term “combination therapy” as used herein means two or moresubstances are administered to a subject over a period of time,contemporaneously or sequentially e.g. the substances can for example beadministered at the same time or at different times within the period oftime, at similar or different intervals. The compounds may or may not bebiologically active in the subject at the same time. As an example, afirst substance is administered weekly, and a second substanceadministered every other week for a number of weeks. The exact detailsof the administration will depend on the pharmacokinetics of the twosubstances. Designs of suitable dosing regimens are routine for oneskilled in the art.

The term “pharmaceutically acceptable” means compatible with thetreatment of animals, in particular, humans.

The term “synergistic” as used herein means the enhanced or magnifiedeffect of a combination on at least one property compared to theadditive individual effects of each component of the combination. Forexample, compounds that induce cell death by the same mechanism, e.g.binding tubulin at the same site would not be expected to have more thanadditive effect. Synergism can be assessed and quantified for example byanalyzing the Data by the Calcusyn median effect model where thecombination index (CI) indicates synergism (CI<0.9), additively(CI=0.9-1.1) or antagonism (CI>1.1). CIs of <0.3, 0.3-0.7, 0.7-0.85,0.85-0.90, 0.90-1.10 or >1.10 indicate strong synergism, synergism,moderate synergism, slight synergism, additive effect or antagonism,respectively. The CI is the statistical measure of synergy.

The term “drug resistant” as used herein refers to a cancer or cancercell, particularly a hematological malignancy or hematological cancercell that resists the effects of chemotherapy, e.g. that is able tosurvive in the presence of a concentration of drug compared to asuitable comparator cell. A drug resistant cancer or cancer cell forexample can require increased concentration of drug for the drug to beeffective. A cell or cancer can become resistant to a drug. For example,a cancer cell can mutate, amplify a gene that renders the drugineffective, pump the drug out of the cell for example viaP-glycoprotein (Pgp), inactivate the drug, prevent transport of the druginto the cell or prevent the cell from dying after exposure to the drug.

In the following passages, different aspects are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

Moreover, all percentages are based on weight percentages unlessotherwise specified. Further, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferences unless the content clearly dictates otherwise. Thus forexample, a composition containing “a compound” includes a mixture of twoor more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Finally, terms of degree such as “substantially”, “about”and “approximately” as used herein mean a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. These terms of degree should be construed as including adeviation of at least ±5% of the modified term if this deviation wouldnot negate the meaning of the word it modifies.

Finally, all references cited herein are incorporated by reference.

II. METHODS/USES

Therapeutic regimens for treating hematological malignancies are hereindescribed.

Flubendazole is demonstrated herein to be cytotoxic to leukemic andmyeloma cells, and to have anti-tumor effects in vivo without causingweight loss, behavioural changes or gross organ toxicity in mice.Accordingly, an aspect of the disclosure includes a method of treating ahematological malignancy in a subject in need thereof comprisingadministering to the subject an effective amount of flubendazole and/ora pharmaceutically acceptable solvate and/or prodrug thereof.

Combination therapies are also disclosed. Accordingly, another aspect ofthe disclosure includes a method of treating a hematological malignancyin a subject in need thereof comprising administering to the subject aneffective amount of flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof, and an effective amount of a vincaalkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof.

Also included in an aspect of the disclosure is a use of flubendazoleand/or a pharmaceutically acceptable solvate and/or prodrug thereof fortreating a hematological malignancy.

Yet another aspect includes a use of flubendazole and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof, incombination with a vinca alkaloid and/or a pharmaceutically acceptablesalt, solvate and/or prodrug thereof for treating a hematologicalmalignancy.

A further aspect includes a use of flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof for themanufacture of a medicament for treating a hematological malignancy.Another aspect includes a use of flubendazole and/or a pharmaceuticallyacceptable solvate and/or prodrug thereof, in combination with a vincaalkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof for the manufacture of a medicament for treating ahematological malignancy.

Another aspect of the disclosure relates to a method of inducing celldeath of a hematological cancer cell comprising contacting the cancercell with an effective amount of flubendazole and/or a pharmaceuticallyacceptable solvate and/or prodrug thereof alone or in combination with avinca alkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof.

Also included is a method of inhibiting tubulin polymerization in a cellcomprising contacting a cell with an effective amount of flubendazoleand/or a solvate and/or prodrug thereof, alone or in combination with avinca alkaloid and/or a salt, solvate and/or prodrug thereof. Inhibitingtubulin polymerization means reducing polymerized tubulin in a cell orsample by at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 95% compared to a suitable control, such as anuntreated cell or sample as determined using assays known in the art.

In an embodiment, the flubendazole is administered before the vincaalkaloid. In another embodiment, the flubendazole is administered afterthe vinca alkaloid. In yet another embodiment, the flubendazole isadministered simultaneously with the vinca alkaloid. In yet anotherembodiment, the flubendazole is administered contemporaneously with thevinca alkaloid. In embodiments, the compounds are administered in asingle dose or in multiple applications, at similar or differentintervals, for example flubendazole is administered daily and a vincaalkaloid is administered once or twice weekly for a particular number orweeks.

In an embodiment, the vinca alkaloid is selected from vinblastine,vincristine, vindesine, and vinolrebine and pharmaceutically acceptablesalts, solvates and prodrugs thereof and mixtures thereof.

In an embodiment, the pharmaceutically acceptable salt of the vincaalkaloid is a sulfate salt or a tartrate salt.

In an embodiment, the hematological malignancy or hematological cancercell is drug resistant to a vinca alkaloid. In a further embodiment, thehematological malignancy or hematological cancer cell overexpressesP-glycoprotein (Pgp). In an embodiment, the method comprises firstidentifying subjects having a hematological malignancy resistant to avinca alkaloid and then administering to the subject an effective amountof flubendazole and/or a pharmaceutically acceptable solvate and/orprodrug thereof, and optionally an effective amount of a vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof. For example, a subject with a hematological malignancy who hasfailed treatment with a vinca alkaloid such as vinblastine orvincristine is identified as a subject having a hematological malignancyresistant to a vinca alkaloid. As an alternative example, a sample ofthe hematological malignancy and/or a cancer cell is tested for drugresistance, for example vinca alkaloid resistance.

Vinca alkaloids such as vincristine are currently used in the treatmentof leukemia and myeloma however neurotoxicity is a dose limitingtoxicity of vincristine. As flubendazole strongly synergized with vincaalkaloids in cell and animal studies it is expected that the sameanti-tumor effect can be obtained with lower concentrations of the vincaalkaloids when combined with flubendazole and/or the combination wouldprovide better anti-tumor efficacy without increased toxicity.

Accordingly, an aspect includes a method of reducing toxicity associatedwith the administration of a vinca alkaloid comprising administering aneffective amount of flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof in combination with a vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof, wherein the quantity of the vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereofadministered is reduced compared to a standard treatment protocol.

Standard treatment protocols for vinca alkaloids are well known in theart. For example, vinblastine can be administered between about 3.7mg/m² bsa and about 18.5 mg/m² bsa and vincristine can be administeredat about 1.4 mg/m² bsa or about up to 2 mg/m² bsa.

The reduction can for example be about 5%, 10%, 15%, 20%, 25%, 30% ormore reduction.

A further aspect includes a method of increasing anti-tumor efficacy ofa vinca alkaloid comprising administering an effective amount offlubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof in combination with a vinca alkaloid and/or a pharmaceuticallyacceptable salt, solvate and/or prodrug thereof.

In another embodiment, the hematological malignancy is a leukemia. Inanother embodiment, the leukemia is selected from acute myeloid leukemia(AML), acute lymphocytic leukemia (ALL) and chronic myeloma leukemia(CML). In an embodiment, the hematological cancer cell is leukemic cell,an AML cell, an ALL cell or a CML cell.

In a further embodiment, the hematological malignancy is a myeloma. Inanother embodiment, the hematological cancer cell is a myeloma cell.

In yet a further embodiment, the hematological malignancy is a lymphoma.In an embodiment, the hematological cancer cell is a lymphoma cell.

In yet a further aspect, the disclosure includes methods and useswherein the compound and/or compounds administered are selected fromflubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof alone or in combination with a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof, andare administered at a dosage and/or comprised dosage form describedherein, for example an oral dosage form or an intravenous dosage form.

III. COMPOSITIONS, COMBINATION PRODUCTS AND KITS

Compositions for treating hematological malignancies are hereinprovided.

An aspect of the disclosure includes a composition comprising aneffective amount of flubendazole, and/or a pharmaceutically acceptableprodrug and/or or solvate thereof, and a suitable carrier or vehicle,for treating hematological malignancies.

Another aspect of the disclosure includes a composition comprising aflubendazole, and/or a pharmaceutically acceptable prodrug and/orsolvate thereof; a vinca alkaloid and/or a pharmaceutically acceptablesalt, prodrug and/or solvate thereof; and, optionally, a suitablecarrier or vehicle.

In an embodiment, the vinca alkaloid is selected from vincristine,vinblastine, vindesine and vinorelbine, and pharmaceutically acceptablesalts, solvates and prodrugs thereof and combinations thereof.

In an embodiment, the pharmaceutically acceptable salt of the vincaalkaloid is a sulfate salt or a tartrate salt.

Another aspect of the disclosure includes a composition comprising aneffective amount of flubendazole, and/or a pharmaceutically acceptableprodrug and/or solvate thereof; a vinca alkaloid and/or apharmaceutically acceptable salt, prodrug and/or solvate thereof; and asuitable carrier or vehicle for treating a hematological malignancy.

The compounds are suitably formulated into pharmaceutical compositionsfor administration to human subjects in a biologically compatible formsuitable for administration in vivo.

The disclosure in an aspect, also describes a pharmaceutical compositioncomprising an effective amount of flubendazole, and/or apharmaceutically acceptable prodrug and/or solvate thereof; a vincaalkaloid and/or a pharmaceutically acceptable salt, prodrug and/orsolvate thereof; and, optionally, a pharmaceutically acceptable carrier,for treatment of a leukemia, lymphoma and/or multiple myeloma.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle.

Suitable vehicles are described, for example, in Remington'sPharmaceutical Sciences (2003-20^(th) Edition). On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which optionally further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that are optionally present in such compositions include, forexample, water, surfactants (such as Tween™), alcohols, polyols,glycerin and vegetable oils. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, tablets, orconcentrated solutions or suspensions. The composition may be supplied,for example but not by way of limitation, as a lyophilized powder whichis reconstituted with sterile water or saline prior to administration tothe subject.

Suitable pharmaceutically acceptable carriers include essentiallychemically inert and nontoxic compositions that do not interfere withthe effectiveness of the biological activity of the pharmaceuticalcomposition. Examples of suitable pharmaceutical carriers include, butare not limited to, water, saline solutions, glycerol solutions,ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride(DOTMA), diolesyl-phosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound(s), together with a suitable amount of carrier so as to providethe form for direct administration to the subject.

In an embodiment, the compositions described herein are administered forexample, by parenteral, intravenous, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol ororal administration.

Compositions for nasal administration may conveniently be formulated asaerosols, drops, gels and powders. Aerosol formulations typicallycomprise a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant which can be a compressed gassuch as compressed air or an organic propellant such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

Wherein the route of administration is oral, the dosage form may be forexample, incorporated with excipient and used in the form of entericcoated tablets, caplets, gelcaps, capsules, ingestible tablets, buccaltablets, troches, elixirs, suspensions, syrups, wafers, and the like.The oral dosage form may be solid or liquid.

Accordingly, a further aspect of the disclosure is a compositionformulated for as an oral dosage form selected from enteric coatedtablets, caplets, gelcaps, and capsules, each unit dosage formcomprising about 10 to less than about 5000 mg, suitably about 10 toabout 3500 mg, about 10 to about 1500 mg, about 10 to about 1200 mg,about 10 to about 1000 mg, about 10 to about 800 mg, about 10 to about500 mg, about 10 to about 300 mg, about 50 to about 3500 mg, about 50 toabout 1500 mg, about 50 to about 1200 mg, about 50 to about 1000 mg,about 50 to about 800 mg, about 50 to about 500 mg, about 50 to about300 mg, about 30 to about 300 mg, or about 35 to about 50 mg, of one ormore compounds selected from flubendazole and/or a pharmaceuticallyacceptable solvate and/or prodrug thereof and/or a vinca alkaloid and/ora pharmaceutically acceptable salt, solvate and/or prodrug thereof and apharmaceutically acceptable carrier.

In an embodiment, the disclosure describes a pharmaceutical compositionwherein the dosage form is a solid dosage form. A solid dosage formrefers to individually coated tablets, capsules, granules or othernon-liquid dosage forms suitable for oral administration. It is to beunderstood that the solid dosage form includes, but is not limited to,modified release, for example immediate release and timed-release,formulations. Examples of modified-release formulations include, forexample, sustained-release (SR), extended-release (ER, XR, or XL),time-release or timed-release, controlled-release (CR), orcontinuous-release (CR or Contin), employed, for example, in the form ofa coated tablet, an osmotic delivery device, a coated capsule, amicroencapsulated microsphere, an agglomerated particle, e.g., as ofmolecular sieving type particles, or, a fine hollow permeable fiberbundle, or chopped hollow permeable fibers, agglomerated or held in afibrous packet. Timed-release compositions can be formulated, e.g.liposomes or those wherein the active compound is protected withdifferentially degradable coatings, such as by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry the compoundsof the disclosure and use the lyophilizates obtained, for example, forthe preparation of products for injection.

Accordingly, a further aspect of the disclosure is a pharmaceuticalcomposition in solid dosage form comprising about 10 to less than about5000 mg, suitably about 10 to about 3500 mg, about 10 to about 1500 mg,about 10 to about 1200 mg, about 10 to about 1000 mg, about 10 to about800 mg, about 10 to about 500 mg, about 10 to about 300 mg, about 50 toabout 3500 mg, about 50 to about 1500 mg, about 50 to about 1200 mg,about 50 to about 1000 mg, about 50 to about 800 mg, about 50 to about500 mg, about 50 to about 300 mg, about 30 to about 300 mg, or about 35to about 50 mg, of one or more compounds selected from flubendazoleand/or a pharmaceutically acceptable solvate and/or prodrug thereofand/or a vinca alkaloid and/or a pharmaceutically acceptable salt,solvate and/or prodrug thereof and a pharmaceutically acceptablecarrier.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, wherein the active ingredient isformulated with a carrier such as sugar, acacia, tragacanth, and/orgelatin and/or glycerin.

In another embodiment, the disclosure describes a pharmaceuticalcomposition wherein the dosage form is a liquid oral dosage form. Aperson skilled in the art would know how to prepare suitableformulations. Conventional procedures and ingredients for the selectionand preparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003-20th edition) and in TheUnited States Pharmacopeia The National Formulary (USP 24 NF19)published in 1999.

Accordingly, a further aspect of the disclosure is a pharmaceuticalcomposition in oral liquid dosage form comprising about 10 to less thanabout 5000 mg, suitably about 10 to about 3500 mg, about 10 to about1500 mg, about 10 to about 1200 mg, about 10 to about 1000 mg, about 10to about 800 mg, about 10 to about 500 mg, about 10 to about 300 mg,about 50 to about 3500 mg, about 50 to about 1500 mg, about 50 to about1200 mg, about 50 to about 1000 mg, about 50 to about 800 mg, about 50to about 500 mg, about 50 to about 300 mg, about 30 to about 300 mg, orabout 35 to about 50 mg, of one or more compounds selected fromflubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof and/or a vinca alkaloid and/or a pharmaceutically acceptablesalt, solvate and/or prodrug thereof and a pharmaceutically acceptablecarrier.

In another embodiment, the disclosure describes a pharmaceuticalcomposition wherein the dosage form is an injectable dosage form. Aninjectable dosage form is to be understood to refer to liquid dosageforms suitable for, but not limited to, intravenous, subcutaneous,intramuscular, or intraperitoneal administration. Solutions of compoundsdescribed herein can be prepared in water suitably mixed with asurfactant such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, DMSO and mixturesthereof with or without alcohol, and in oils. Under ordinary conditionsof storage and use, these preparations contain a preservative to preventthe growth of microorganisms. A person skilled in the art would know howto prepare suitable formulations. Conventional procedures andingredients for the selection and preparation of suitable formulationsare described, for example, in Remington's Pharmaceutical Sciences(2003-20th edition) and in The United States Pharmacopeia: The NationalFormulary (USP 24 NF19) published in 1999.

Accordingly, a further aspect of the disclosure is a pharmaceuticalcomposition in injectable dosage form comprising about 10 to less thanabout 5000 mg, suitably about 10 to about 3500 mg, about 10 to about1500 mg, about 10 to about 1200 mg, about 10 to about 1000 mg, about 10to about 800 mg, about 10 to about 500 mg, about 10 to about 300 mg,about 50 to about 3500 mg, about 50 to about 1500 mg, about 50 to about1200 mg, about 50 to about 1000 mg, about 50 to about 800 mg, about 50to about 500 mg, about 50 to about 300 mg, about 30 to about 300 mg, orabout 35 to about 50 mg, of one or more compounds selected fromflubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof and/or a vinca alkaloid and/or a pharmaceutically acceptablesalt, solvate and/or prodrug thereof and a pharmaceutically acceptablecarrier.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

In an embodiment, the dosage form comprises about 10 mg to about 5000 mgof one or more compounds selected from flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof. In anotherembodiment, the dosage form comprises about 10 mg to about 1500 mg ofone or more compounds selected from flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof. In yetanother embodiment, the dosage form comprises about 30 mg to about 300mg of one or more compounds selected from flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof. In otherembodiments, the dosage form comprises about 10 to about 3500 mg, about10 to about 1200 mg, about 10 to about 1000 mg, about 10 to about 800mg, about 10 to about 500 mg, about 10 to about 300 mg, about 50 toabout 3500 mg, about 50 to about 1500 mg, about 50 to about 1200 mg,about 50 to about 1000 mg, about 50 to about 800 mg, about 50 to about500 mg, about 50 to about 300 mg, about 30 to about 300 mg, or about 35to about 50 mg, of one or more of one or more compounds selected fromflubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof.

In one embodiment the dosage, for example the daily dosage, comprisesabout 20 mg to about 5000 mg of one or more compounds described herein.In another embodiment, the dosage comprises about 100 mg to about 1500mg of one or more compounds described herein. In yet another embodiment,the dosage comprises about 400 to about 1200 mg of one or more compoundsdescribed herein. In other embodiments, the dosage form comprises about10 to about 3500 mg, about, about 10 to about 1200 mg, about 10 to about1000 mg, about 10 to about 800 mg, about 10 to about 500 mg, about 10 toabout 300 mg, about 50 to about 3500 mg, about 50 to about 1500 mg,about 50 to about 1200 mg, about 50 to about 1000 mg, about 50 to about800 mg, about 50 to about 500 mg, about 50 to about 300 mg, about 30 toabout 300 mg, or about 35 to about 50 mg of one or more compoundsdescribed herein.

The dosage form may alternatively comprise about 1 to about 200 mg ofone or more compounds described herein/kg body weight, about 2 to about100 mg of one or more compounds described herein/kg body weight, about20 to about 100 mg of one or more compounds described herein/kg bodyweight, about 5 to about 50 mg of one or more compounds describedherein/kg body weight about 5 to about 25 mg of one or more compoundsdescribed herein/kg body weight, or about 5 to about 15 mg of one ormore compounds described herein/kg body weight of a subject in need ofsuch treatment formulated into a solid oral dosage form, a liquid oraldosage form, or an injectable dosage form. In an embodiment, the dosagecomprises about 5 to about 15 mg of one or more compounds describedherein/kg body weight of a subject in need of such treatment formulatedinto a solid oral dosage form, a liquid dosage oral form, or aninjectable dosage form.

In an embodiment, the dosage form comprises an effective amount or atherapeutically effective amount. In one embodiment the dosage formcomprises about 10 to about 5000 mg of one or more compounds describedherein. In another embodiment, the dosage form comprises about 10 toabout 1500 mg of the compound(s). In another embodiment, the dosage formcomprises about 30 to about 300 mg of the compound(s).

The flubendazole and/or a pharmaceutically acceptable solvate and/orprodrug thereof and the vinca alkaloid and/or a pharmaceuticallyacceptable salt, solvate and/or prodrug thereof are optionally in thesame dosage form or in different dosage forms. For example flubendazoleis optionally in an oral dosage form and the vinca alkaloid is aninjectable dosage form.

Also included in another aspect of the disclosure, is a kit comprisingflubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof and a vinca alkaloid and/or a pharmaceutically acceptable salt,solvate and/or prodrug thereof for treating a hematological malignancy.

Another embodiment includes a kit comprising flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof andinstructions for use in combination with a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof fortreating a hematological malignancy.

Another embodiment includes a kit comprising a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof andinstructions for use in combination with flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof for treatinga hematological malignancy.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

To date, the identification of drugs with unanticipated anti-cancereffects has been largely serendipitous. Disclosed herein is a systematicapproach to identify compounds with unanticipated anti-cancer activityby testing libraries of drugs in chemical screens. From these screens,flubendazole, a member of the benzimidazole family of antihelminticdrugs, was identified to have anti-leukemia and anti-myeloma activity.Flubendazole has been extensively evaluated in humans and animals forthe treatment of intestinal parasites as well as for the treatment ofsystemic worm infections. In these studies, patients have received up to50 mg/kg orally daily for 24 months without serious adverseeffects.¹²⁻¹⁴ Healthy volunteers have also received single oral doses upto 2000 mg without toxicity.¹⁵ In sheep receiving a single flubendazoledose of 5 mg/kg intravenously, no toxicity was observed and the areaunder the curve (AUC) was 6.53 μg·h/mL (22 μM).¹⁶

While selected members of the benzimidazole family have recently beenreported to induce cell death in solid tumor cell lines,¹⁷⁻¹⁹ theanti-tumor properties of flubendazole have not been previously reported.Moreover, the mechanism by which benzimidazoles exert their effects asantihelmintics and by which they induce cell death in malignant cells isnot fully understood and several cellular responses have been described.For example, benzimidazoles have been shown to inhibit amino peptidaseactivity and glutamate catabolism, reduce glucose uptake, increaseintracellular calcium levels, and inhibit microtubule formation.²⁰⁻²²

Flubendazole displays anti-leukemia and anti-myeloma activity in vitroand in vivo at pharmacologically achievable concentrations.Mechanistically, flubendazole inhibits tubulin structure and function byinteracting with a site on tubulin distinct from vinca alkaloid tubulininhibitors. In keeping with this different mechanism of tubulininhibition, flubendazole synergized with vinca alkaloids, vinblastineand vincristine to induce cell death and delay tumor growth in vivo.Therefore, given its prior safety and toxicity testing, flubendazolecould be rapidly repurposed as a novel agent for the treatment ofleukemia and myeloma for use in combination with vinca alkaloids.

Results

A Screen of Drugs for Compounds with Novel Anti-Cancer ActivityIdentifies Flubendazole

To identify drugs with unanticipated anti-cancer activity, a library of110 drugs focused on anti-microbials and metabolic regulators with awide therapeutic index and well characterized pharmacokinetics wascompiled. This library was then screened to identify compounds that werecytotoxic to leukemia cell lines³⁴ and identified several cytotoxicagents including mebendazole. Mebendazole is a member of thebenzimidazole family of antihelmintics, so the cytotoxicity of this drugclass was investigated. OCI-AML2 leukemia cells were treated withincreasing concentrations of 8 benzimidazoles. Seventy two hours afterincubation, cell growth and viability was measured by the MTS assay. Themost potent benzimidazole in this panel was flubendazole (FIG. 1A).

Flubendazole is Cytotoxic to Leukemia and Myeloma Cell Lines

Having identified flubendazole as a potential anti-cancer agent, itseffects were evaluated in a panel of malignant cell lines. Leukemia andmyeloma cell lines were treated with increasing concentrations offlubendazole. Seventy two hours after incubation, cell growth andviability was measured by the MTS assay. Flubendazole reduced cellviability with an LD₅₀≦1.0 μM in 8/8 myeloma and 4/6 leukemia celllines, including MDAY-D2 cells with an LD₅₀ of 3.0 nM (FIG. 1B). Ofnote, the remaining 2 cells lines (OCI-AML-2 and CEM) had LD₅₀ values of1.07±0.6 and 1.9±0.9 μM respectively. Concentrations of 1.0 μM appearpharmacologically achievable, based on prior studies that demonstratedan AUC of 22 μM in the absence of toxicity.¹⁶ Cell death was confirmedby PI staining and the trypan blue exclusion assay. Flubendazole alsoreduced the clonogenic growth of primary AML samples (FIG. 1C). Thus,flubendazole displays activity against leukemia and myeloma cells atnanomolar concentrations.

Flubendazole Delays Tumor in Leukemia and Myeloma Xenografts

As flubendazole was cytotoxic to malignant cells in vitro, itsanti-tumor effects were evaluated in leukemia and myeloma xenografts.OCI-AML2 leukemia cells were injected subcutaneously into sub-lethallyirradiated SCID mice. Mice were then treated with flubendazole (20 or 50mg/kg) daily or vehicle control intraperitoneally. Compared to micetreated with vehicle control, flubendazole significantly delayed tumorgrowth and reduced tumor weights (p<0.0001; FIGS. 2A-B). Likewise,sub-lethally irradiated SCID mice were injected subcutaneously with OPM2myeloma cells. Mice were then treated daily with 50 mg/kg offlubendazole or buffer control for 17 days intraperitoneally.Flubendazole significantly delayed tumor growth and reduced tumorweights compared to mice treated with vehicle control (p<0.05; FIG. 2C).

In both leukemia and myeloma models, there was no evidence of weightloss, behavioral changes or gross organ toxicity with flubendazoletreatment. Thus, flubendazole displays novel pre-clinical activityagainst leukemia and myeloma.

Flubendazole does not Alter Glucose Uptake

In studies with the parasite Trichuris globulosa, the antihelminticeffects of the benzimidazoles thiabendazole and fenbendazole wererelated to inhibition of glucose uptake with resultant alterations inglucose metabolism.²¹ Therefore, we tested the effects of flubendazoleon glucose uptake in malignant cells. OCI-AML2 cells were treated withincreasing concentrations of flubendazole for 16 hours and uptake of3H-deoxy-D-glucose was measured (FIG. 6A). In contrast to theobservations in the parasite, flubendazole at concentrations up to 4.0μM did not alter glucose uptake. Likewise, culturing cells with varyingconcentrations of glucose did not alter flubendazole-induced death (FIG.6B). Thus, flubendazole-induced cell death does not appear related toinhibition of glucose uptake.

Flubendazole Alters Microtubule Structure and Function

To better understand the mechanism by which flubendazole induced deathof malignant cells changes in gene expression following 4 hours offlubendazole treatment were examined. By gene ontology and pathwayanalysis, 196 genes were identified to be deregulated >4 fold withflubendazole treatment (ArrayExpress accession: E-MEXP-2352; Table 2).Of these, 179 genes were annotated and 58/179 fell within 8 functionalannotations associated with chromosomal segregation and cytoskeletonregulation; genes involved in chromosomal segregation were the mostaffected by flubendazole treatment (Table 3). Moreover, by ConnectivityMap analysis, changes in gene expression were found to be similar togene signatures induced by the known tubulin inhibitors nocodazole andcolchicine.

The effects of flubendazole on tubulin structure and polymerization wereevaluated. To determine whether flubendazole alters tubulin structure,changes in the number of reactive cysteine residues on tubulin followingincubation with flubendazole were measured. Treatment with flubendazolereduced the number of reactive cysteines by 5.2±2.6 compared to buffercontrol (p<0.05). In comparison, vinblastine decreased the number ofreactive cysteines by 8.6±2.5 (p<0.01). Thus, these results suggest thatflubendazole interacts with tubulin to alter its structure (FIG. 3A).

Drugs that alter microtubules can either promote or inhibit tubulinpolymerization.³⁵ Therefore, the effects of flubendazole on tubulinpolymerization were investigated. Flubendazole was incubated withpurified bovine tubulin and tubulin polymerization was recorded overtime. As controls, bovine tubulin was incubated with colchicine which isknown to inhibit tubulin polymerization³⁶ and taxol which is known topromote tubulin polymerization.³⁷ In this assay, flubendazole inhibitedtubulin polymerization (FIG. 3B).

As flubendazole altered the structure and polymerization of tubulin, itsbinding site to vinblastine, a member of the vinca alkaloid family ofmicrotubule inhibitors that is currently used for the treatment ofleukemia and myeloma was compared.^(38,39) Purified bovine tubulin wasincubated with flubendazole or vinblastine followed by the addition ofcolchicine. The interaction of colchicine with tubulin was then assayedby measuring the fluorescence of colchicine bound to tubulin.⁴⁰ Theaddition of flubendazole decreased colchicine fluorescence, but theaddition of vinblastine had no effect (FIG. 3C), indicating thatflubendazole, but not vinblastine, blocks colchicine from bindingtubulin. Thus flubendazole interacts with tubulin through a mechanismdistinct from vinblastine.

Since flubendazole altered tubulin structure and function in cell-freeassays, the effects of flubendazole on microtubule formation in intactcells were evaluated. PPC-1 prostate cancer cells were treated withflubendazole (1.0 μM) or vehicle control for 24 hours, and then stainedwith anti-tubulin and DAPI. Microtubule architecture was visualized byconfocal microscopy (FIG. 3D). PPC-1 cells treated with vehicle controlexhibited an organized network of elongated microtubules (FIG. 3Di). Incontrast, cells treated with flubendazole were rounded with contractedand disorganized microtubules (FIG. 3Dii). Thus, flubendazole disruptsthe microtubule architecture in intact cells.

Microtubules mediate cell migration⁴¹, the effects of flubendazole oncell migration with a wound healing assay were investigated. HeLa cellswere seeded in 4 well chambers and after adhering overnight, the cellmonolayer was scratched to create a wound. Cells were treated withflubendazole or buffer control and migration of cells to heal the woundwas measured overtime. Treatment with flubendazole impaired cellmigration and delayed wound healing (FIG. 3E). Of note, at theconcentrations and times tested in this these assays, theflubendazole-treated cells were more than 89% viable as measured by MTSassay.

Flubendazole Arrests Cells in Cell Cycle and Induces Mitotic Catastrophe

Inhibition of tubulin polymerization can inhibit cell cycle progressionand induce mitotic catastrophe⁴², so the effects of flubendazole on thecell cycle by flow cytometry and on chromosomal segregation byenumerating the number of multi-nucleated cells in OCI-AML2 and PPC-1cells were assessed. Flubendazole arrested cells in the G2 phase of thecell cycle (FIGS. 4A-B) and increased the number of multi-nucleatedcells (FIG. 4C). Thus, flubendazole produces cell cycle arrest andmitotic catastrophe, consistent with its effects as a microtubuleinhibitor.

Inhibition of Microtubules is Functionally Important for Flubendazole'sCytotoxicity

Next, whether microtubule inhibition was functionally important forflubendazole's cytotoxicity was determined. KB-4.0-HTI cells have asingle nucleotide change in α-tubulin that renders it resistant totubulin inhibitors.²³ Therefore, we treated KB-4.0-HTI and KB-3-1 wildtype controls with increasing concentrations of flubendazole andcolchicine. Consistent with flubendazole's effects on tubulin,KB-4.0-HTI cells were more resistant to flubendazole with an IC₅₀ 7-foldhigher than the non-mutated KB-3-1 wild type cells (Table 1). Similarly,KB-4.0-HTI cells were also resistant to colchicine with an IC₅₀ 2.5-foldhigher than the KB-3-1 control cells (Table 1), consistent with previousobservations with this cell line.²³ Flubendazole was also tested inA549.EpoB40 cells, which are more sensitive to microtubule inhibitorsdue to a point mutation in β-tubulin at residue 292.²⁴ A549.EpoB40 cellswere more sensitive to flubendazole with an IC₅₀ 5-fold lower than thenon-mutated A549 control cells (Table 1). Similarly, A549.EpoB40 cellswere sensitive to colchicine with an IC₅₀ 1.5-fold lower than the A549control cells (Table 1), consistent with previous observations with thiscell line.²⁴ As further evidence that flubendazole's cytotoxicity wasrelated to microtubule inhibition, the benzimidazoles thiabendazole and1,3-benzidiazole that did not induce cell death in OCI-AML2 cells (FIG.1A) also did not inhibit microtubule formation in our cell-freepolymerization assays. Thus, taken together, flubendazole induces celldeath through a mechanism that appears related to its inhibition ofmicrotubule polymerization.

Over-Expression of P-Glycoprotein does not Alter Flubendazole'sCytotoxicity.

Over-expression of P-glycoprotein (Pgp, MDR1) renders cells resistant tovinca alkaloid microtubule inhibitors.⁴³ Therefore, the effects of Pgpover-expression on flubendazole's cytotoxicity were tested. CEM-VBLcells over-express Pgp,⁴⁴ which was confirmed with a rhodamine dyeexclusion assay. CEM wild type and CEM-VBL cells were treated withincreasing concentrations of flubendazole or vinblastine and cellviability measured by the MTS assay (Table 1). CEM-VBL cells remainedfully sensitive to flubendazole compared to the wild type controls. Incontrast, CEM-VBL cells over-expressing Pgp were resistant tovinblastine at concentrations greater than 5.0 μM (Table 1). Therefore,over-expression of Pgp does not abrogate the cytotoxicity offlubendazole and this drug is capable of over-coming some forms ofresistance to vinca alkaloids.

Flubendazole does not Induce Neuropathy

Neuropathy is a dose limiting toxicity of vinca alkaloid microtubuleinhibitors such as vincristine. Therefore, the effects of flubendazoleon neurologic function in mice were tested. Mice (n=10/group) weretreated with 50, 100, and 200 mg/kg of flubendazole or vehicle controlintraperitoneally daily for 14 days, and sensory function was assessedwith the tail-flick assay. No changes in tail-flick latency wereobserved at doses up to 200 mg/kg compared to controls, a dose 10-foldhigher than the dose required for an anti-tumor effect (mean±SDtail-flick latency: control 3.04±0.52 seconds vs. 200 mg/kg flubendazole2.91±0.50 seconds, p=0.58 by Student's t-test). However, doses of 200mg/kg resulted in 5% decrease in body weight compared to vehiclecontrols (p=0.03, Student's t-test).

Flubendazole Synergizes with Vinblastine and Enhances Vinblastine andVincristine Activity In Vivo

Flubendazole interacts with tubulin through a mechanism distinct fromvinblastine. Therefore, the cytotoxicity of flubendazole and vinblastinein combination was evaluated. OCI-AML2 cells were treated withincreasing concentrations of flubendazole and vinblastine and 72 h afterincubation cell growth and viability was measured by the MTS assay.Combinations were assessed based on CI values where CI values <1, equalto 1 or >1 are considered synergistic, additive or antagonistic,respectively.^(33,45) The combination of flubendazole and vinblastinesynergistically induced cell death with CI values of 0.09, 0.017, 0.003and 0.001 at the EC 50, 25, 10 and 5, respectively (FIG. 5A). Incontrast, cell death produced by the combination of flubendazole andcolchicine was closer to additive with CI values of 0.54, 0.70, 0.897and 1.07 at EC 50, 25, 10 and 5, respectively (FIG. 5B).

Given the synergy of flubendazole and vinblastine in cell culture, thecombination of flubendazole and vinblastine and vincristine in vivo wasevaluated. OCI-AML2 cells were injected subcutaneously into SCID miceand treated intraperitoneally with flubendazole (15 mg/kg), vinblastine(0.3 mg/kg) or vincristine (0.25 mg/kg or 0.35 mg/kg), or thecombination of the two agents. The combination of flubendazole andvinblastine decreased tumor weight greater than either agent alone(p<0.01) (FIG. 5C). Similarly, the combination of flubendazole andvincristine decreased tumor weight greater than either agent alone(p<0.001) (FIG. 5D). Moreover, there were no observed behavioralchanges, weight loss, or gross organ toxicity from either combinationtreatment (FIG. 7). Thus, flubendazole synergizes with vinblastine andvincristine and could be used in combination with these vinca alkaloidsto achieve a greater anti-tumor effect.

Discussion

Through screens of libraries of drugs flubendazole was identified ashaving previously unrecognized anti-leukemia and anti-myeloma activity.At pharmacologically achievable concentrations, flubendazole inducedcell death in malignant cells and delayed tumor growth in vivo.Mechanistically, flubendazole altered microtubule structure andinhibited tubulin polymerization by interacting with a site on tubulinsimilar to colchicine and distinct from vinblastine.

As part of its development as an antihelmintic, flubendazole has beenstudied extensively in animals and humans, where it has displayedfavorable toxicology profiles. For example, in rats, mice andguinea-pigs the LD₅₀ is >5000 mg/kg and >400 mg/kg after oral andintraperitoneal administration, respectively.¹⁵ No toxicity was noted inrats that received up to 150 mg/kg/day for 3 months, while chickensreceiving up to 180 mg/kg flubendazole daily for 7 days developed anemiaand reduction of red cells in the spleen.¹⁵ In humans, doses of 40-50mg/kg/day for 10 days have been administered for the treatment ofneurocysticercosis and no toxicity from the drug was reported.¹⁴Likewise, patients received up to 50 mg/kg/day of flubendazole for up to24 months for the treatment of alveolar echinococcosis without adverseeffect.¹³

The pharmacokinetics of flubendazole are also well characterized. Forexample, in sheep the estimated half life for flubendazole after oraladministration is 6.5 hours and the main metabolic pathways arecarbamate hydrolysis and ketone reduction.¹⁵ After intravenousadministration, an AUC of 22 μM is achieved over 36 hours after a singleintravenous dose of 5 mg/kg. However, only 18% of flubendazole isabsorbed, so after a single oral dose of 5 mg/kg the AUC over 36 hourswas 1.17 μg·h/mL (4.0 μM).¹⁶ Similar pharmacokinetics are observed inhealthy human volunteers and patients receiving flubendazole for thetreatment of parasitic infection. For example, in healthy volunteers whoreceived 1000 mg flubendazole as a single oral dose, 77% of unchangeddrug was detected in the feces and less than 0.1% in the urine threedays after administration.¹⁵ Thus, taken together, flubendazole has poororal bioavailability. However, since large doses can be safelyadministered and since oral administration of flubendazole has clinicalefficacy in the treatment of systemic worm infections, oral flubendazolewould be expected to be useful for anti-cancer activity. Alternatively,an intravenous formulation would be useful.

In support of the evaluation of flubendazole for the treatment ofpatients with refractory hematologic malignancies, a Phase I clinicaltrial of the related benzimidazole, albendazole was recently conductedin patients with advanced solid tumors.⁴⁶ In 2 of 7 patients,albendazole reduced levels of the tumor markers AFP and CEA. However,albendazole caused severe neutropenia in 3 of these patients and thedevelopment of albendazole as an anti-tumor agent has not been pursuedto date.

In the present study, flubendazole inhibited tubulin polymerization andfunction which were functionally important for its cytotoxicity.Microtubules are cytoskeleton components that are required for celldivision, cellular transport and in the maintenance of cellularintegrity.³⁵ Microtubules are comprised of α- and β-tubulin heterodimersthat assemble into linear protofilaments and polymerize into hollow,cylindrical structures.³⁸ The gain or loss of tubulin heterodimers leadsto elongation or shortening of the microtubules.⁴⁷ Drugs that alter thepolymerization of microtubule are well validated therapeutic agents forthe treatment of malignancies. For example, vinca alkaloids are used inthe treatment of leukemia and myeloma and inhibit microtubulepolymerization by binding to β-tubulin near the GTP-binding site.³⁸ Incontrast, colchicine inhibits polymerization by binding the interface ofthe α/β-tubulin heterodimer and taxol promotes tubulin polymerization bybinding in the lumen of the polymer.³⁸ In our study, we demonstratedthat flubendazole interacts with tubulin at a site similar to that ofcolchicine and distinct from vinca-alkaloids. A similar interaction withtubulin has been reported for the benzimidazole mebendazole.⁴⁸ However,the benzimidazole benomyl has been reported to inhibit tubulinpolymerization by interacting at a site distinct from both thecolchicine site and the vinca domain.¹⁹ Thus, the mechanism by whichbenzimidazoles inhibit tubulin formation appears to vary among familymembers.

Vinca alkaloids are currently used in the treatment of leukemia andmyeloma, and neurotoxicity is a dose limiting toxicity of vincristine.In contrast, flubendazole did not produce acute neurotoxicity asevaluated in the tail-flick assay. However, additional neurotoxicologytesting with more prolonged dosing could fully evaluate the sensory andmotor effects of this drug. It is noted that prior animal toxicologystudies and human clinical trials have not reported neurotoxicity afterflubendazole administration.

As flubendazole and vinca alkaloids inhibited tubulin through distinctmechanisms, the combination of these drugs was evaluated. Flubendazolesynergized with the vinca alkaloids vinblastine and vincristine in vitroand in vivo. Therefore, these drugs can be used in combination toenhance the efficacy of standard therapy for these diseases orpotentially reduce their toxicity.

Finally, as flubendazole inhibits tubulin through a mechanism distinctfrom vinca alkaloids, flubendazole can be useful in overcoming someforms of resistance to vinca alkaloids. For example, as over-expressionof Pgp does not render cells resistant to flubendazole, it could alsoovercome this specific mechanism of resistance to vinca alkaloids.

In summary, flubendazole is a novel inhibitor of microtubules that actsthrough a mechanism distinct from vinca alkaloids. Given its priorsafety record in humans and animals coupled with its pre-clinicalefficacy in hematological malignancies, flubendazole can be repurposedfor evaluation in these diseases.

Materials and Methods Reagents

Colchicine, taxol, and the panel of benzimidazole compounds werepurchased from Sigma Chemical (St. Louis, Mo.). Vinblastine waspurchased from Calbiochem (San Diego, Calif.). Drugs were prepared asstock solutions in dimethyl sulfoxide (DMSO).

Cell Culture

Leukemia (U937, MDAY, CEM, CEM-VBL) and solid tumor cell lines (PPC-1,HeLa) were cultured in RPMI 1640 medium. The epidermoid carcinoma celllines KB-3-1 and KB-4.0-HTI²³ were grown in Dulbecco's Modified EagleMedium. The lung carcinoma cell lines A549 and A549.EpoB40 were grown inRPMI with the latter supplemented with 20% fetal calf serum (FCS), and40 nM epothilone B.²⁴ Leukemia cell lines OCI-AML2, CEM, and NB4 and allthe myeloma cell lines (OPM2, KMS11, JJN3 LP1, H929, L1, KMS12, KSM18and OCI My5) were maintained in Iscove's Modified Dulbecco's Medium(IMDM). TEX cells were maintained in IMDM, 15% FCS, 1%penicillin-streptomycin, 2 mM L-glutamine, 20 ng/mL SCF, 2 ng/mL IL-3.Unless otherwise noted, all media were supplemented with 10% FCS, 100units/mL of streptomycin and 100 μg/mL of penicillin (all from Hyclone,Logan, Utah). Cells were incubated in a humidified air atmospherecontaining 5% CO₂ at 37° C.

Primary human acute myeloid leukemia (AML) samples were isolated fromthe peripheral blood of consenting AML patients, who had at least 80%malignant cells among the mononuclear cells in their peripheral bloodand cultured at 37° C. in IMDM, 10% fetal calf serum, 100 units/mL ofstreptomycin and 100 μg/mL of penicillin. The collection and use ofhuman tissue for this study was approved by the local ethics reviewboard (University Health Network, Toronto, ON, Canada).

Cell Growth and Viability Assays

Cell growth and viability was measured using the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuminner salt (MTS) reduction assay (Promega, Madison, Wis.) according tothe manufacture's protocol and as previously described²⁵. Cells wereseeded in 96 well plates and treated with drug for 72 h. Optical density(OD) was measured at 490 nm.

Mitotic catastrophe was measured by enumerating the number ofmultinucleated cells similar to the method previously described²⁶.Briefly, cells (5.0×10⁵) were fixed and stained as described below.Fields containing 100 cells per treatment group were selected at randomand visualized at 20×. A total of 700 cells were scored per treatmentcondition. A positive score for mitotic catastrophe was given when twoor more distinct nuclear lobes were visualized within a single cell.

Analysis of Gene Expression

Changes in gene expression were measured in U937 leukemia cells treatedwith 1.0 μM flubendazole or buffer control for 4 hours using IngenuityPathways Analysis (www.ingenuity.com) and the Database for Annotation,Visualization and Integrated Discovery (DAVID;http://david.abcc.ncifcrf.gov). Gene expression was measured in U937leukemia cells treated with 1.0 μM flubendazole or buffer control for 4hours. Briefly, total RNA was harvested from treated and untreated cellsand hybridized to Affymetrix HG U133 Plus 2.0 gene expressionoligonucleotide arrays (Affymetrix, Santa Clara, Calif., USA). Labelingand hybridization to arrays were performed by The Centre for AppliedGenomics (Medical and Related Sciences Centre, Toronto, ON, Canada).Microarray data were analyzed using GeneSpring GX v10.0 (Agilent), andgenes deregulated >4-fold after 4 hours flubendazole treatment wereidentified. Pathways and gene ontology analyses were carried out usingIngenuity Pathways Analysis (www.ingenuity.com) and the Database forAnnotation, Visualization and Integrated Discovery (DAVID;http://david.abcc.ncifcrf.gov). Connectivity Map analysis, whichcompares the changes in gene expression signature following flubendazoletreatment with gene expression signatures following treatment with othercommon pharmacological agents, was also performed(http://www.broadinstitute.org/cmap).

Leukemia and Myeloma Xenograft Models

Sub-lethally irradiated SCID mice were injected subcutaneously in theleft flank with leukemia OCI-AML2 (2.0×10⁶) or myeloma OPM2 (1.0×10⁷)cells. Mice were then randomly assigned to receive flubendazole (in 0.9%NaCl and 0.01% tween-80) or vehicle control (0.9% NaCl and 0.01%tween-80) intraperitoneally. When the combination of flubendazole andvinblastine or vincristine was evaluated, mice were randomly assigned toreceive flubendazole (in 0.9% NaCl and 0.01% tween-80), vinblastine (inPBS and 0.01% tween-20) or vincristine (in PBS and 0.01% tween-20), thecombination of flubendazole and vinblastine or vincristine, or vehiclecontrol intraperitoneally. Tumor volumes (tumor length×width²×0.5236)were monitored daily using calipers. At the end of the experiment (16-18days), mice were sacrificed, tumors excised and tumor volume and weightmeasured. All animal studies were carried out according to theregulations of the Canadian Council on Animal Care and with the approvalof the local ethics review board.

Assessment of Glucose Uptake

The effect of flubendazole on the uptake of 2-deoxy-D-glucose inOCI-AML2 cells was performed using a radioactive glucose uptake assay asdescribed by Wood et al²⁷. OCI-AML2 cells were treated with increasingconcentrations of flubendazole or the known glucose uptake inhibitor,cytochalasin B. After treatment, cells were washed twice with aHEPES-buffered saline solution (140 mM NaCl, 5 mM KCl, 2.5 mM MgSO₄, 1mM CaCl₂, and 20 mM Hepes, pH 7.4). Uptake of 2-deoxy-D-glucose wasinitiated by the addition of 10 μM 2-deoxy-D-glucose and 1 μCi/mL2-[³H]deoxy-D-glucose in the HEPES-buffered saline solution at roomtemperature. After 5 min of incubation, 2-deoxy-D-glucose uptake wasterminated by rinsing the cells three times with a 25 mM ice-coldglucose solution, followed by cell disruption with 0.05 N NaOH. Proteinconcentration was measured by the modified Bradford protein assay andcell-associated radioactivity was determined by scintillation counting.The mean counts per minute are expressed as picomoles/min/mg protein.

Tubulin Polymerization Assay

Polymerization of bovine tubulin was measured according to Beyer etal²⁸. Briefly, bovine tubulin (1.8 mg/ml; Cytoskeleton; Denver, USA) wasadded to ice cold polymerization buffer (PEM; 80 mM PIPES, 0.5 mM EGTA,2 mM MgCl₂, 10% glycerol and 1 mM GTP) and centrifuged at top speed in amicrocentrifuge for 5 min at 4° C. Supernatant (100 μL/well) wasimmediately added to a 96 well plate, which contained colchicine, taxol,flubendazole, or buffer/DMSO control in PEM buffer. Final concentrationsof colchicine, taxol and flubendazole were 6.0 μM, 6.0 μM and 100 μM,respectively. Following addition of tubulin, the plate was immediatelyplaced in the spectrophotometer, which was maintained at 37° C., and theabsorbance measured every 3 minutes for 2.5 h at 340 nm.

Measurement of Tubulin Sulfhydryl Groups

The ability of microtubule target drugs to alter tubulin structure wasassessed by measuring the number of reactive cysteine residues using thesulfhydryl reagent 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB; Sigma) aspreviously described^(19,29). Briefly, bovine tubulin (1.5 μM) wasincubated with 10 μM vinblastine, 10 μM colchicine, 100 μM flubendazoleor a control for 15 min at 4° C. After incubation, DTNB was added (100μM final concentration) and the absorbance was measured in a 1 cm pathlength cuvette at 412 nm for 60 min at 37° C. The number of sulfhydrylgroups were determined by using a molar extinction coefficient for DTNBof 13 600 (M⁻¹, cm⁻¹).²⁹

Determining the Site of Binding to Tubulin

The site of flubendazole binding was determined similar to Gupta etal¹⁹. Bovine tubulin (5.0 μM) was incubated with 100 μM flubendazole,100 μM vinblastine or buffer control for 30 min at 37° C. Colchicine (10μM) was then added and incubated for an additional 60 min at 37° C.Fluorescence of the colchicine-tubulin complex was subsequently measuredat excitation and emission wavelengths of 360 nm and 430 nm,respectively.

Determining Effect of Flubendazole Microtubules in Cultured Cells

PPC-1 cells (5.0×10³) were seeded on glass cover slips in a 6 well plateovernight at 37° C. and then treated with 1.0 μM flubendazole or vehiclecontrol for 24 h. Cells were then fixed in 3.7% paraformeldehyde for 15min at 37° C., permeabilized using 0.1% triton X-100 for 4 min at roomtemperature, and incubated at 4° C. overnight in a humidified chamberwith a blocking solution (0.5% BSA). Cells were then incubated with aprimary mouse anti α-tubulin antibody (clone DMIA, 1:300, in 0.5% BSA;Sigma) for 1 hour at 37° C., washed 3 times in PBS and subsequentlyincubated with a goat anti-mouse IgG, AlexaFluor 488 secondary antibody(1:500, Sigma) for 1 hour in the dark at room temperature. Cells werethen washed, stained with 4′,6-diamidino-2-phenylindole (DAPI) andmounted on a microscope slide using Dakocytomation fluorescent mountingmedia (Dakocytamation, Carpinteria, Calif.). Slides were imaged usingthe Olympus Fluoview FV1000 Laser Scanning Confocal Microscope (OlympusAmerica Inc., Center Valley, Pa.) at room temperature at 40×.

Cell Migration Assays

HeLa cell migration was measured as previously described³⁰. Briefly,HeLa cells were seeded in 4 well chambers and grown to confluence. Awound was inflicted to the monolayer using a 200 μL pipette tip. Wellswere washed with media to remove any detached cells and media containingeither 0 μM, 0.5 μM or 1.0 μM flubendazole was then added and incubatedat 37° C. for 8 hours. Images were captured using a Zeiss Axiovert 200Mmicroscope every 2 hours. These images were quantified using Image Jsoftware, which outlined and enumerated the number of pixels in thewound area. Cell migration of HeLa cells was recorded and calculated asa percent recovery of wound area.

Cell Cycle Analysis

Cell cycle analysis was performed as described by Mao et al³¹. Briefly,OCI-AML2 or PPC-1 cells were harvested, washed with cold PBS andresuspended in PBS and cold absolute ethanol. Cells were then treatedwith 100 ng/mL of DNase-free RNase A (Invitrogen, Carlsbad, Calif.) at37° C. for 30 min, washed with cold PBS, resuspended in PBS andincubated with 50 μg/mL of propidium iodine (PI) for 15 min at roomtemperature in the dark. DNA content was measured by flow cytometry(FACSCalibur, Becton Dickinson, Florida, USA) and analyzed with FlowJosoftware version 7.0 (TreeStar, Ashland, Oreg.).

Assessment of Sensory Function with the Tail-Flick Assay

Sensory function was assessed with the tail-flick assay by ChempartnersCo. (Shanghai, China) similar to previously described³². Briefly, 50experimentally-naïve, male, adult C57BU6J mice from Shanghai SLAC Co.Ltd were treated with 50, 100, 200 mg/kg of flubendazole in 0.9% NaCland 0.01% tween-80 or vehicle control (n=10 per group). Before and after14 days of treatment, tail flick latency was measured by applying ahigh-intensity, noxious, radiant heat stimulus 20 mm from the tip of thetail. When a withdrawal tail-flick response occurred, the thermalstimulus was terminated automatically and the response latency wasmeasured electronically.

Drug Combination Studies

The combination index (CI) was used to evaluate the interaction betweenflubendazole and colchicine or vinblastine. OCI-AML2 cells were treatedwith increasing concentrations of flubendazole, vinblastine orcolchicine. Seventy-two hours after incubation cell viability wasmeasured by the MTS assay. The Calcusyn median effect model was used tocalculate the CI values and evaluate whether the combination offlubendazole with vinblastine or colchicine was synergistic,antagonistic or additive. CI values of <1 indicate synergism, CI=1indicate additivity and Cl>1 indicate antagonism.³³

Statistical Analysis

Unless otherwise stated, the results are presented as mean±SD. Data wereanalyzed using GraphPad Prism 4.0 (GraphPad Software, USA). p<0.05 wasaccepted as being statistically significant. Drug combination data wereanalyzed using Calcusyn software (Biosoft, UK).

TABLE 1 Flubendazole effects on mutated cell lines. Cell lineFlubendazole (μM) Colchicine (μM) KB-3-1 1.9 ± 1.1 14.9 ± 4.5 KB-4.0-HTI 12.5 ± 1.8  41.6 ± 5.5  A549 4.1 ± 1.3 0.09 ± 0.01A549.EpoB40 0.8 ± 0.2 0.06 ± 0.01 Flubendazole (μM) Vinblastine (μM) CEM1.9 ± 0.9 0.14 ± 0.01 CEM-VBL 2.7 ± 1.2 >5.0 Data represent IC₅₀ values,as measured by the MTS assay, calculated from 3 replicates.

TABLE 2 Gene pathway analysis. Fold- Difference Deregu- Gene Symbol orProbe set ID (4 hr) lation Unigene ID Name 228196_s_at 4.039798 downHs.631814 transcribed locus 211307_s_at 4.047831 down Hs.659872 FCAR211363_s_at 4.050233 down Hs.193268 MTAP 241666_at 4.088215 downHs.55131 C3orf23 229676_at 4.108218 down Hs.173946 PAPD1 215043_s_at4.125255 down Hs.652536 SMA4 SMA5 211087_x_at 4.140346 down Hs.588289MAPK14 1557918_s_at 4.168854 down Hs.75231 SLC16A1 209761_s_at 4.178591down Hs.145150 SP110 1554767_s_at 4.188646 down Hs.352671 CRYZL1222614_at 4.204624 down Hs.34136 RWDD2B 221194_s_at 4.217304 downHs.531701 RNFT1 1552703_s_at 4.232619 down Hs.348365 CASP1 COP1231918_s_at 4.241984 down Hs.277154 GFM2 242288_s_at 4.257984 downHs.532815 EMILIN2 203622_s_at 4.348037 down Hs.262858 PNO1 1558015_s_at4.413946 down Hs.699451 ACTR2 1552486_s_at 4.455582 down Hs.410388 LACTB1558775_s_at 4.467875 down Hs.372000 NSMAF 210180_s_at 4.50081 downHs.533122 SFRS10 1553530_a_at 4.503789 down Hs.695946 ITGB1 1555814_a_at4.505738 down Hs.247077 RHOA 212142_at 4.618687 down Hs.460184 MCM4226825_s_at 4.627178 down Hs.479766 TMEM165 1553940_a_at 4.633946 downHs.578684 LRRC28 223925_s_at 4.737739 down Hs.602015 LOC100130332LOC100134793 LUZP6 MTPN 76897_s_at 4.767624 down Hs.522351 FKBP15206116_s_at 5.055958 down Hs.133892 TPM1 232675_s_at 5.136206 downHs.504998 UCKL1 204427_s_at 5.145536 down Hs.75914 TMED2 227364_at5.153504 down 220494_s_at 5.194894 down 215073_s_at 5.237517 downHs.701977 NR2F2 229835_s_at 5.263865 down Hs.656865 SLMO2 208748_s_at5.305004 down Hs.179986 FLOT1 207495_at 5.334993 down Hs.656060 RAB281554424_at 5.341551 down Hs.518760 FIP1L1 207419_s_at 5.382367 downHs.517601 RAC2 210935_s_at 5.491971 down Hs.128548 WDR1 226570_at5.504646 down Hs.477789 ATP1B3 227299_at 5.520281 down Hs.709250 CCNI223341_s_at 5.524223 down Hs.480815 SCOC 203372_s_at 5.566165 downHs.485572 SOCS2 1554768_a_at 5.570022 down Hs.591697 MAD2L1 219858_s_at5.710344 down Hs.708489 FLJ20160 216521_s_at 5.722223 down Hs.558537BRCC3 205461_at 5.779909 down Hs.524788 RAB35 212009_s_at 5.792535 downHs.337295 STIP1 201946_s_at 5.829165 down Hs.189772 CCT2 241733_at5.868862 down Hs.208701 C18orf54 219599_at 5.968014 down Hs.648394 EIF4B229212_at 6.051483 down Hs.644056 transcribed locus 233878_s_at 6.079963down Hs.255932 XRN2 221276_s_at 6.246901 down Hs.712631 SYNC1202226_s_at 6.256392 down Hs.638121 CRK 231972_at 6.439062 downHs.656166 CDNA: FLJ21028 fis, clone CAE07155 1555154_a_at 6.482312 downHs.510324 QKI 242277_at 6.48723 down Hs.701344 transcribed locus212105_s_at 6.670136 down Hs.191518 DHX9 224407_s_at 6.680334 downHs.444247 RP6-213H19.1 227658_s_at 6.693072 down Hs.41086 PLEKHA3215719_x_at 6.719481 down Hs.244139 FAS 213562_s_at 6.760106 downHs.71465 SQLE 216252_x_at 6.859726 down Hs.244139 FAS 223143_s_at 7.051down Hs.485915 C6orf166 209535_s_at 7.296443 down 217294_s_at 7.459872down Hs.517145 ENO1 219927_at 7.737007 down Hs.579828 FCF1 244187_at7.950194 down Hs.648463 transcribed locus 231370_at 8.033558 downHs.707434 transcribed locus 204426_at 8.122973 down Hs.75914 TMED2229787_s_at 8.148592 down Hs.705676 transcribed locus 227260_at 8.706609down Hs.525163 ANKRD10 1565717_s_at 8.748013 down Hs.513522 FUS211102_s_at 9.204724 down Hs.655593 LILRA2 243495_s_at 9.238962 downHs.712948 MRNA; cDNA DKFZp686E18224 222611_s_at 9.446891 down Hs.213198PSPC1 229713_at 9.591681 down Hs.644929 transcribed locus 238474_at9.751438 down Hs.510375 NUP43 228746_s_at 10.68661 down Hs.518265transcribed locus 229128_s_at 10.89904 down Hs.656466 transcribed locus230659_at 11.50984 down Hs.673002 transcribed locus 1555193_a_at11.88252 down Hs.655904 ZNF277 AFFX- 17.0748 down HUMRGE/M1 0098_5_at230265_at 17.40556 down Hs.181300 SEL1L 231576_at 17.47066 downHs.702816 transcribed locus 203032_s_at 19.22807 down Hs.592490 FH213872_at 27.33301 down Hs.592644 transcribed locus 232148_at 4.004654up Hs.372000 NSMAF 243768_at 4.01657 up Hs.696546 transcribed locus243158_at 4.025012 up 230998_at 4.031707 up Hs.653966 transcribed locus241893_at 4.051749 up Hs.655355 transcribed locus 242837_at 4.085199 upHs.469970 SFRS4 1559117_at 4.101708 up Hs.634052 CDNA FLJ34664 fis,clone LIVER2000592 244427_at 4.101731 up Hs.270845 KIF23 237388_at4.122815 up Hs.49105 GLMN 237768_x_at 4.129501 up 1562511_at 4.136996 upHs.532411 LYST 229871_at 4.157612 up Hs.612332 SAMD4B 230712_at 4.158728up Hs.655246 KIAA1245 LOC100132406 NBPF1 NBPF10 NBPF11 NBPF14 NBPF15NBPF16 NBPF20 NBPF3 NBPF8 NBPF9 RP3-377D14.1 XXyac-YX155B6.1 215191_at4.161385 up Hs.636888 CDNA FLJ14085 fis, clone HEMBB1002534 1556338_at4.162479 up Hs.662144 CDNA FLJ39845 fis, clone SPLEN2014452 229514_at4.181521 up Hs.410231 C14orf118 234723_x_at 4.183379 up Hs.677287 CDNA:FLJ21228 fis, clone COL00739 234032_at 4.186586 up Hs.684536 transcribedlocus 234989_at 4.195736 up TncRNA 232597_x_at 4.208668 up Hs.210367SFRS2IP 230651_at 4.216365 up Hs.666664 transcribed locus 230099_at4.217873 up Hs.605074 transcribed locus 243303_at 4.220261 up 1558710_at4.230851 up Hs.687438 CDNA FLJ40669 fis, clone THYMU2020883 244015_at4.26262 up 217164_at 4.266195 up 240247_at 4.27683 up 242261_at 4.299726up 242476_at 4.309448 up Hs.605126 transcribed locus 1558467_a_at4.323054 up Hs.656444 Clone IMAGE: 125405, mRNA sequence 232094_at4.359978 up Hs.633566 C15orf29 1560145_at 4.360516 up Hs.709388 MKLN1239091_at 4.379727 up Hs.659152 transcribed locus 229422_at 4.417276 upHs.584782 NRD1 216211_at 4.425798 up Hs.659130 MRNA; cDNA DKFZp564A023(from clone DKFZp564A023) 1559490_at 4.449507 up Hs.518414 LRCH31553349_at 4.464808 up Hs.696080 ARID2 243527_at 4.476238 up 240008_at4.490335 up Hs.656290 transcribed locus 215204_at 4.501104 up Hs.661961CDNA FLJ14090 fis, clone MAMMA1000264 242431_at 4.504952 up 233300_at4.538653 up Hs.660696 CDNA FU11548 fis, clone HEMBA1002944 234148_at4.56567 up Hs.677340 CDNA: FLJ21585 fis, clone COL06903 238558_at4.60338 up Hs.674068 transcribed locus 232889_at 4.617942 up GUSBP11562062_at 4.670624 up Hs.655246 KIAA1245 LOC100132406 NBPF1 NBPF10NBPF11 NBPF14 NBPF15 NBPF16 NBPF20 NBPF3 NBPF8 NBPF9 RP3-377D14.1XXyac-YX155B6.1 229483_at 4.685478 up Hs.657369 CDNA FLJ42331 fis, cloneTSTOM2000588 221899_at 4.689089 up Hs.507680 N4BP2L2 243037_at 4.723519up 237632_at 4.724212 up 244061_at 4.799224 up 213700_s_at 4.800273 upHs.655868 transcribed locus 232529_at 4.812432 up Hs.531587 SP3223494_at 4.876228 up Hs.500842 MGEA5 238735_at 4.877998 up 201694_s_at4.88721 up Hs.326035 EGR1 235023_at 4.917645 up Hs.511668 VPS13C1562194_at 4.956551 up Hs.684712 Full length inser

cDNA clone YW28D08 243964_at 4.956945 up Hs.605805 transcribed locus228105_at 4.965934 up Hs.655980 transcribed locus 241775_at 4.987032 upHs.662113 CDNA FLJ26437 fis, clone KDN02067 242233_at 5.029235 upHs.665440 transcribed locus 242558_at 5.068929 up Hs.658202 CDNAFLJ45490 fis, clone BRTHA2005831 235716_at 5.075923 up Hs.569031transcribed locus 1563130_a_at 5.101688 up Hs.661154 MRNA full lengthinsert cDNA clone EUROIMAGE 626063 242673_at 5.143738 up Hs.599613transcribed locus 227576_at 5.161981 up Hs.276976 CDNA FLJ42015 fis,clone SPLEN2032813 242121_at 5.171345 up Hs.653288 RNF12 239243_at5.176998 up Hs.434401 ZNF638 239784_at 5.187452 up Hs.669403 transcribedlocus 206115_at 5.250307 up Hs.534313 EGR3 239937_at 5.284644 upHs.706835 ZNF207 228723_at 5.290023 up Hs.656678 CDNA FLJ30445 fis,clone BRACE2009238 235757_at 5.328636 up Hs.675679 transcribed locus231281_at 5.335745 up 235138_at 5.36946 up Hs.662054 transcribed locus1565703_at 5.505036 up Hs.75862 SMAD4 230292_at 5.506033 up Hs.709358LOC100131993 230494_at 5.543806 up Hs.187946 SLC20A1 242008_at 5.598142up Hs.671336 transcribed locus 230387_at 5.76847 up Hs.659630transcribed locus 1569181_x_at 5.881034 up Hs.662486 transcribed locus215269_at 6.028463 up Hs.126221 TMEM1 1557543_at 6.033797 up Hs.684024transcribed locus 1565886_at 6.471508 up Hs.621480 Full length inser

cDNA clone ZB94A08 1569180_at 6.566242 up Hs.662486 transcribed locus233271_at 6.631978 up Hs.677062 CDNA FLJ11709 fis, clone HEMBA10051331559156_at 6.635394 up Hs.658076 CDNA clone IMAGE: 5266106 215611_at6.663847 up Hs.511504 TCF12 242068_at 6.670528 up Hs.603603 transcribedlocus 217591_at 6.680076 up Hs.677805 transcribed locus 1557527_at6.746184 up Hs.675708 CDNA FLJ33848 fis, clone CTONG2005567 207746_at6.833061 up Hs.241517 POLQ 235847_at 6.946846 up Hs.661841 transcribedlocus 243827_at 7.25954 up Hs.601123 transcribed locus 239516_at7.304538 up Hs.125291 transcribed locus 241681_at 7.460481 up Hs.656858transcribed locus 239597_at 7.573097 up 1556590_s_at 7.724134 upHs.633049 CDNA FLJ25645 fis, clone SYN00113 236114_at 7.796954 upHs.666463 transcribed locus 203665_at 7.974161 up Hs.517581 HMOX1235879_at 8.350311 up Hs.478000 MBNL1 243149_at 8.513012 up Hs.669156transcribed locus 230332_at 9.280883 up Hs.654700 ZCCHC7 222371_at9.376632 up Hs.675666 transcribed locus 235925_at 9.392304 up Hs.511504TCF12 236561_at 13.18963 up Hs.494622 TGFBR1 225239_at 16.84248 upHs.593027 CDNA FLJ26120 fis, clone SYN00419

indicates data missing or illegible when filed

TABLE 3 Gene ontology analysis. Fold Enrich- Term Count % P-value Genesment BP00206: 12 6.70% 0.0019 244427_at 3.057853 Chromosome 1554768_a_atsegregation 230292_at 206116_s_at 210935_s_at 241775_at 243303_at1553530_a_at 1558015_s_at 1562062_at 239597_at 229514_at BP00032: 105.59% 0.011 243158_at 2.746405 Purine 242121_at metabolism 231576_at206116_s_at 1553530_a_at 227260_at 216211_at 235757_at 211363_s_at239597_at 242068_at BP00001: 9 5.03% 0.038 213700_s_at 2.352284Carbohydrate 215043_s_at metabolism 215043_s_at 243303_at 217294_s_at230659_at 232889_at 1554767_s_at 1563130_a_at 242261_at BP00101: 9 5.03%0.022 219599_at 2.617163 Sulfur 230998_at metabolism 215719_x_at216252_x_at 1562062_at 230712_at 206116_s_at 217294_s_at 244015_at243149_at 1563130_a_at 242261_at 239516_at BP00281: 8 4.47% 0.022232675_s_at 2.853996 Oncogenesis 237768_x_at 231576_at 206116_s_at1565717_s_at 211307_s_at 1562062_at 215191_at BP00180: 8 4.47% 0.0098206115_at 3.376069 Detoxification 215043_s_at 230265_at 242008_at1562062_at 230712_at 1554424_at 236561_at 232529_at BP00114: 8 4.47%0.0001 242121_at 5.385946 MAPKKK 229835_s_at cascade 206116_s_at230659_at 238474_at 230651_at 239243_at 229514_at BP00005: 7 3.91% 0.016213700_s_at 3.450612 Glycolysis 1556590_s_at 228105_at 228196_s_at243303_at 217294_s_at 242288_s_at 1562062_at BP00033: 7 3.91% 0.011229128_s_at 3.773099 Pyrimidine 232675_s_at metabolism 216521_s_at235023_at 1555154_a_a 211307_s_at 1554767_s_at BP00129: 6 3.35% 0.040229128_s_at 3.184482 Endocytosis 223341_s_at 230659_at 238474_at211087_x_at 232529_at

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent disclosure wasspecifically and individually indicated to be incorporated by referencein its entirety.

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

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1. A method of treating a hematological malignancy in a subject in needthereof comprising administering to the subject an effective amount offlubendazole and/or a pharmaceutically acceptable solvate and/or prodrugthereof.
 2. The method of claim 1 comprising administering to thesubject an effective amount of flubendazole and/or a pharmaceuticallyacceptable solvate and/or prodrug thereof, and an effective amount of avinca alkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof.
 3. The method of claim 2, wherein the flubendazoleand/or a pharmaceutically acceptable solvate and/or prodrug thereof isadministered before, simultaneously with, or after the vinca alkaloidand/or a pharmaceutically acceptable salt, solvate and/or prodrugthereof.
 4. The method of claim 2, wherein the vinca alkaloid isselected from vinblastine, vincristine, vindesine and vinolrebine and apharmaceutically acceptable salt, solvate and/or prodrug thereof.
 5. Themethod of claim 1, wherein the pharmaceutically acceptable salt is asulfate salt or a tartrate salt.
 6. The method of claim 1, wherein thehematological malignancy is drug resistant to a vinca alkaloid and/oroverexpresses Pgp.
 7. The method of claim 1, wherein the hematologicalmalignancy is leukemia, lymphoma or myeloma.
 8. The method of claim 7,wherein the leukemia is AML, ALL or CML.
 9. (canceled)
 10. The method ofclaim 1, wherein the flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof and/or the vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof arecomprised in a single oral dosage form or separate oral dosage forms.11. The method of claim 1, wherein the flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof and/or thevinca alkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof are comprised in a single intravenous dosage form orseparate intravenous dosage forms.
 12. A composition comprising aneffective amount of flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof and a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof. 13.The composition of claim 12, wherein the vinca alkaloid is selected fromvincristine, vinblastine, vindesine and vinorelbine and apharmaceutically acceptable salt, solvate and prodrug thereof.
 14. Thecomposition of claim 12, wherein the pharmaceutically acceptable salt isa sulfate salt or a tartrate salt.
 15. The composition of claim 12,wherein the composition is for treating a hematological malignancy.16.-28. (canceled)
 29. A kit comprising flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof andoptionally a vinca alkaloid and/or a pharmaceutically acceptable salt,solvate and/or prodrug thereof for treating a hematological malignancyaccording to the method of claim
 1. 30. A kit comprising flubendazoleand/or a pharmaceutically acceptable solvate and/or prodrug thereof andinstructions for use in combination with a vinca alkaloid and/or apharmaceutically acceptable salt, solvate and/or prodrug thereof fortreating a hematological malignancy according to the method of claim 1.31. A kit comprising a vinca alkaloid and/or a pharmaceuticallyacceptable salt, solvate and/or prodrug thereof and instructions for usein combination with flubendazole and/or a pharmaceutically acceptablesolvate and/or prodrug thereof for treating a hematological malignancyaccording to the method of claim
 1. 32. A method according to claim 1for reducing toxicity associated with the administration of a vincaalkaloid comprising administering an effective amount of flubendazoleand/or a pharmaceutically acceptable solvate and/or prodrug thereof incombination with a vinca alkaloid and/or a pharmaceutically acceptablesalt, solvate and/or prodrug thereof, wherein the quantity of the vincaalkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof administered is reduced compared to a standard treatmentprotocol.
 33. The method of claim 32 wherein the quantity of vincaalkaloid and/or a pharmaceutically acceptable salt, solvate and/orprodrug thereof is reduce by about 5%, 10%, 15%, 20%, 25%, 30% or more.34. A method of increasing anti-tumor efficacy of a vinca alkaloidcomprising administering an effective amount of flubendazole and/or apharmaceutically acceptable solvate and/or prodrug thereof incombination with a vinca alkaloid and/or a pharmaceutically acceptablesalt, solvate and/or prodrug thereof.